Methods for making a microfluidic aliquot chip

ABSTRACT

According to the invention, generally, a method for making a microfluidic aliquoting (MA) chip, adapted to fit in a Petri dish, has a center well (inlet) connected by branched channels to a plurality of side wells (outlets). The chip comes in various types, including a bMA Chip T1, bMA Chip T2, bMA Chip T3, and an rMA Chip. The branched channel improvement provides for a greater distance between neighboring channels and a decreased density near the center well. Design improvements including an injection mold design for an insert and a base and a multiplex hole punch allow for rapid fabrication of the MA chip.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation in part of U.S. applicationSer. No. 15/701,401 filed Sep. 11, 2017, which is a continuation of U.S.application Ser. No. 15/006,634 filed Jan. 26, 2016, now issued as U.S.Pat. No. 9,757,728 which are each hereby incorporated herein byreference in their respective entirety.

TECHNICAL FIELD

The present invention, in some embodiments thereof, relates broadly tomethods and apparatus for single-cell isolation (such as for processingor analysis) and, more particularly to techniques for isolating singlecells using microfluidic technology.

BACKGROUND

Microfluidics is a multidisciplinary field intersecting engineering,physics, chemistry, biochemistry, nanotechnology, and biotechnology,with practical applications to the design of systems in which lowvolumes of fluids are processed to achieve multiplexing, automation, andhigh-throughput screening. Advances in microfluidics technology arerevolutionizing molecular biology procedures for enzymatic analysis(e.g., glucose and lactate assays), DNA analysis (e.g., polymerase chainreaction and high-throughput sequencing), and proteomics. The basic ideaof microfluidic biochips is to integrate assay operations such asdetection, as well as sample pre-treatment and sample preparation on onechip.

There is increased evidence that phenotypic and genotypic heterogeneityin cell populations widely exists. The key information from individualrare cells may be masked by bulk cell analysis. Single-cell analysis,especially sequencing of DNA and RNA, has therefore become significantlyimportant for clonal mutation, tumor evolution, embryonic development,and immunological intervention.

The initial and key step for such downstream single-cell geneticanalysis is to effectively isolate live single cells of interest fromheterogeneous cell populations into submicroliter medium volume,followed by PCR (polymerase chain reaction) analysis. (PCR is atechnology in molecular biology used to amplify a single copy or a fewcopies of a piece of DNA across several orders of magnitude, generatingthousands to millions of copies of a particular DNA sequence.)

Besides laser capture microdissection primarily used to isolate singlecells from formalin-fixed paraffin-embedded tissue, there are currentlyfour main approaches for single-cell isolation from cell suspensions.The most frequently used method is serial dilution, which has beenwidely applied for colony formation but is not suited for PCR analysiswhere single cells should be isolated into submicroliter suspensions fora better amplification reaction. Even so, these colonies are stillrequired for further analysis to judge if they originate from singlecells which are very difficult to be accurately counted after seedinginto 96-well plates. A second method is micromanipulation, which ismainly developed to isolate single cells for genome/transcriptomesequencing. However, micromanipulation is a time-consuming process andits low throughput feature makes it difficult to rapidly prepare dozensand even hundreds of single cells. Moreover, it is highly dependent on aresearcher's ability to suck single cells. Another method is flowcytometry, which is a well-established method particularly suitable forhigh-throughput sorting of specific cells based on a preset fluorescencegating strategy. However, maintenance of high single-cell viability ischallenging and it doesn't work when only a limited number of cells,such as precious clinical samples, are available.

Due to the comparable dimensions of microchannels and cells,microfluidic technology provides a unique and efficient method forsingle cell manipulation. However, potential disadvantages, includingrequirement of additional skills for microfluid manipulation, poorcompatibility with existing experimental platforms, andinability/difficulty to selectively retrieve the isolated single cellsfrom microchips for further analysis, greatly limit its application incommon laboratories. The original Microfluidic Aliquot Chip (MA-Chip)comprises 120 channels that directly connect to one center inlet well ina radial pattern, resulting in a space of less than 40 μm between twoneighboring channels around the center inlet well. Although the 40 μm offabrication resolution can be well achieved by photolithography to makePDMS MA-Chips, it is challenging to fabricate such MA-Chips with plasticmaterials, such as polystyrene (PS), polypropylene (PP), polycarbonate(PC), and polymethyl methacrylate (PMMA), by using injection molding orlaser cutting. Therefore, an alternative design of MA-Chip with theincreased space of two neighboring channels is required for the massfabrication of plastic MA-Chips.

The branched MA-Chip Type 1 (bMA-Chip T1) in the present invention isdesigned to increase the space between two neighboring channels aroundthe center inlet well while maintaining uniform liquid distribution fromthe center inlet well to the outlet wells. The original MA-Chip containsone segment, in contrast, the bMA-Chip T1 contains multiple segments,allowing the channel number around the center inlet well to decreasefrom 120 to 24 and even 12. These improvements reduce channel densityand provide an extra space around the center inlet well. As a result,the space between neighboring channels increases from less than 40 μm tomore than 400 μm. The improved design in bMA-Chip T1 can meet therequirement for the mass fabrication of plastic MA-Chips.

An objective of the present invention is to provide a technique forsimple, rapid, and versatile single-cell isolation using microfluidictechnology. In this invention, a single cell is isolated by aliquotingfrom a suspension of a large number of cells, independently of cellsize, shape, and motility. The original microfluidic aliquot chipprovides such functions, however it consists of a plurality of straightchannels in the chip, resulting in the densely positioned channels. Thebranched Microfluidic Aliquot Chip-Type 2 (bMA Chip-T2) and the branchedMicrofluidic Aliquot Chip-Type 3 (bMA Chip-T3) in the present inventionare designed as an improvement to the original MA-chip due to themultiple branched channels. This offers the advantage of reducedclogging, strengthened sealing, and enhanced isolation through uniformdistribution of flow resistance into the branched channels.

The original design and fabrication methods of the original MA-Chip arebased on photolithography followed by soft PDMS casting on the mold.While the photolithography based manufacturing process ensures highresolution of the microstructures in MA-Chip, the daily manufacturingoutput is limited. The roughly estimated production cycle time for aMA-Chip is 1 hr. This low production rate results in high productioncosts. To reduce both production costs and final market price, theMA-Chip must be redesigned so that it is appropriate for standard massproduction strategy such as the injection molding process. The newdesign is suitable for a high output production process such asinjection molding to greatly increase the production rate.

The injection mold design of the present MA-Chip allows mass productionby the injection molding process. With standard operation, the estimatedproduction cycle time for a single device is 10 s, which is 360 timeshigher than the original rate. Compared to the original design, the newdesign also improves MA-Chip function, operation, and compatibilities.The device is assembled and packaged for ready to use application. Itreduces additional operations such as placing the MA-Chip on a flatsterile surface and ensuring sealing of the flow channel.

The original MA-Chip has a unique design of radial channels connecting acenter inlet to surrounding outlet wells and provides the capability toisolate and identify rare single cells in a mixed population with asimple pipetting operation. The vital design and fabrication element isthe smooth connection of micrometer scale channels with millimeter scalewells. In the original manufacturing scheme, the micrometer sizechannels (30-80 μm) are fabricated by soft lithography followed by PDMSmolding, and the millimeter scale wells (1.5-2 mm) are created bymechanical punch press. Thus, this manual operation demands asignificant amount of time and labor. To improve throughput of the holepunch process, a multiplexed hole punch device is designed.

A multiplex hole punch strategy for the rapid fabrication of MA-Chip isdesigned to meet the requirement of mass production while maintainingthe original MA-Chip manufacturing format. Current operation requiresthe holes to be punched manually by trained individuals. In one MA-Chip,there are 96-120 holes and alignment is required in each hole punchprocess. The quality of outcome and labor time in this process highlydepends on the operator's skill and experience. The multiplex hole punchis designed to increase throughput of the hole punch process whilemaintaining the original design format of the MA-Chip.

Photolithography is suitable for fabricating high quality PDMS channelsbut difficult for making holes. In contrast, laser cutting or injectionmolding can easily achieve mass production of plastic holes butdifficult to make high quality channels. The two methods can be combinedto achieve rapid fabrication of MA-Chips. The present invention includesa basic strategy for the rapid fabrication of MA-Chip to meet therequirement of mass production. The operation is to align and combinetwo patterned layers.

In the original MA-Chip, the outlet wells are primarily located in theedge of the device with a radial pattern. However, the majority of theMA-Chip is occupied by radial channels, resulting in wasted space anddifficulty in further increasing the number of outlet wells to meet therequirement of high-throughput assay, such as a device containinghundreds to thousands of wells. Therefore, a new design of the MA-Chipis required. The present invention has a rectangular MA-Chip (rMA-Chip)that has the potential to integrate hundreds to thousands of outletwells in the size of a standard 96-well plate.

U.S. Pat. No. 6,632,656 (2003 Oct. 14; Thomas et al.), incorporated byreference herein, discloses apparatus and methods for performing cellgrowth and cell based assays in a liquid medium. The apparatus comprisesa base plate supporting a plurality of micro-channel elements, eachmicro-channel element comprising a cell growth chamber, an inlet channelfor supplying liquid sample thereto and an outlet channel for removal ofliquid sample therefrom, a cover plate positioned over the base plate todefine the chambers and connecting channels, the cover plate beingsupplied with holes to provide access to the channels. Means areincorporated in the cell growth chambers, for cell attachment and cellgrowth. More particularly, as shown and described therein:

-   -   Referring to FIG. 1b, the apparatus comprises a rotatable        disc (18) microfabricated to provide a sample introduction port        located towards the centre of the disc and connected to an        annular sample reservoir (9) which in turn is connected to a        plurality of radially dispersed micro-channel assay elements (6)        each of said micro-channel elements comprising a cell growth        chamber, a sample inlet channel and an outlet channel for        removal of liquid therefrom and a cover plate positioned onto        said disc so as to define closed chambers and connecting        channels. Each micro-channel element is connected at one end to        the central sample reservoir (9) and at the opposing end to a        common waste channel (10).    -   Each of the radially-dispersed micro-channel elements (6) of the        microfabricated apparatus (shown in FIG. 1a) comprises a sample        inlet channel (1) connected at its left hand-end end to the        reservoir (9), a cell growth chamber (2) for performing cell        growth and connected through a channel (4) to an assay        chamber (3) and an outlet channel (5) connected at its        right-hand end to the waste channel (10).    -   Suitably the disc (18) is of a one- or two-piece moulded        construction and is formed of an optionally transparent plastic        or polymeric material by means of separate mouldings which are        assembled together to provide a closed structure with openings        at defined positions to allow loading of the device with liquids        and removal of waste liquids. In the simplest form, the device        is produced as two complementary parts, one or each carrying        moulded structures which, when affixed together, form a series        of interconnected micro-channel elements within the body of a        solid disc. Alternatively the micro-channel elements may be        formed by micro-machining methods in which the micro-channels        and chambers forming the micro-channel elements are        micro-machined into the surface of a disc, and a cover plate,        for example a plastic film, is adhered to the surface so as to        enclose the channels and chambers.    -   The scale of the device will to a certain extent be dictated by        its use, that is the device will be of a size which is        compatible with use with eukaryotic cells. This will impose a        lower limit on any channel designed to allow movement of cells        and will determine the size of cell containment or growth areas        according to the number of cells present in each assay. An        average mammalian cell growing as an adherent culture has an        area of ˜300 μm²; non-adherent cells and non-attached adherent        cells have a spherical diameter of ˜10 μm. Consequently channels        for movement of cells within the device are likely to have        dimensions of the order of 20-30 μm or greater. Sizes of cell        holding areas will depend on the number of cells required to        carry out an assay (the number being determined both by        sensitivity and statistical requirements). It is envisaged that        a typical assay would require a minimum of 500-1000 cells which        for adherent cells would require structures of 150,000-300,000        μm², i.e. circular ‘wells’ of ˜400-600 μm diameter.    -   The configuration of the micro-channels . . . is preferably        chosen to allow simultaneous seeding of the cell growth chamber        by application of a suspension of cells in a fluid medium to the        sample reservoir by means of the sample inlet port, followed by        rotation of the disc (18) by suitable means at a speed        sufficient to cause movement of the cell suspension outward        towards the periphery of the disc by centrifugal force. The        movement of liquid distributes the cell suspension along each of        the inlet micro-channels (1, 8) towards the cell growth chambers        (2, 7). The rotation speed of the disc is chosen provide        sufficient centrifugal force to allow liquid to flow to fill the        cell growth chamber (2, 7), but with insufficient force for        liquid to enter the restricted channel (4, 16) of smaller        diameter on the opposing side of the cell growth chamber.

BRIEF SUMMARY OF THE EMBODIMENTS OF THE INVENTION

In a variant, a microfluidic aliquot (MA) chip for isolating cells,comprises a chip having a center, a top surface, a bottom surface, anouter edge and a thickness. The chip has an inlet well disposedsubstantially at the center of the chip, extending into and accessiblefrom the top surface of the chip. A plurality of outlet wells aredisposed in an outer portion of the chip, extending into and accessiblefrom the top surface of the chip. A plurality of multiple segmentsextend from the inlet well to the outlet wells, wherein the multiplesegments comprise branched channels. The chip is configured to maintainuniform distribution of a liquid and cells from the inlet well to theoutlet wells.

In another variant, the MA chip comprises a plurality of first segmentsthat form an inner section, a plurality of last segments that form anouter section, and a plurality of segments between the first segmentsand the last segments that form a middle section. The first segment isconnected to the center inlet well in a radial pattern and each segmentafter the first segment is divided from a prior segment in a radialpattern. A first channel of a last segment is shorter than a secondchannel of the last segment. Every other outlet well is disposed fartheraway from the inlet well than an adjacent outlet well.

In a further variant, the plurality of segments in the middle sectionform a curved portion generally in the shape of a bend, whereby one ofthe first segments is joined to the bend of a segment in the middlesection.

In yet another variant, the chip comprises four first segments thatextend outward from the inlet well in four cardinal directions,respectively. Each segment branches at a 90 degree angle in two oppositedirections into another segment forming a set of branched channels. Eachset of branched channels on a last segment correspond to a set of outletwells.

In another variant, the chip comprises a plurality of first segmentsthat extend outward radially from the inlet well. Each segment branchesat a 90 degree angle in two opposite directions into another segmentforming a set of branched channels. Each set of branched channels on alast segment correspond to a set of outlet wells.

In a further variant, the chip comprises a sheet of material selectedfrom a group consisting of polydimethylsiloxane (PDMS), PMMA(poly(methyl methacrylate)), PS (polystyrene), and PC (polycarbonate).The sheet has a top surface and a bottom surface, that correspond to thetop and bottom surfaces of the chip, respectively.

In yet another variant, the chip comprises a center inlet well having adiameter of 2-4 mm and a volume of 3-5 μl. A plurality of side outletwells have a diameter of 1.5-2 mm and a volume of 1-3 μl. A plurality ofbranched channels have a width of 50-100 μm. The chip is configured tofit within a Petri dish of 8.5-10 cm.

In another variant, the chip comprises a first layer having a topsurface and a bottom surface and a second membrane layer having a topsurface and a bottom surface. The bottom surface of the second membranelayer has an adhesive and the bottom surface of the second membranelayer is configured to adhere to the top surface of the first layer.

In a further variant, the top surface of the first layer comprises aninlet well, an outlet well array, a main-channel array, and asub-channel array. The main channels are longer and wider than thesub-channels. The main channels extend outward from the inlet well tothe sub-channels and the sub-channels extend from the main channels tothe outlet wells.

In yet another variant, the chip comprises a second membrane layer thathas a thickness of 0.03-0.3 mm. A plurality of outlet wells have adiameter of 0.5-5 mm and a height of 1-10 mm. The chip has a length of127.8±5 mm, a width of 85.5±3 mm, and a height of 1-10 mm.

In another variant, the inlet well comprises a cap having a hole that isconfigured to attach onto the inlet well and receive a cell suspension.

In a further variant, the center inlet well is configured to receive anddistribute liquid through the branched channels and into the outletwells.

In yet another variant, an MA chip insert for making an MA chip,comprises a center through-hole, a plurality of aliquotingthrough-holes, a plurality of branched channels, a plurality of outletwells, a bottom surface, a top surface, a flattened edge, and aplurality of rivet through-holes. The insert has a design configured tomatch a design of an MA chip.

In another variant, an MA chip base for making an MA chip, comprises abottom surface having bottom pillars, a top surface having edge wells, asink plateau surface having a flattened edge, and a plurality of rivets.

In a further variant, a method for making an MA chip, comprisesinserting a first pair of patterned metal injection molds into aninjection molding machine; injecting thermoplastic into the first pairof patterned metal injection molds; demolding to produce an inserthaving a reverse pattern of the first pair of molds; inserting a secondpair of patterned metal injection molds into the injection moldingmachine; inserting thermoplastic into the second pair of patterned metalinjection molds; demolding to produce a base having a reverse pattern ofthe second pair of molds; inserting a plurality of rivets on the baseinto a plurality of rivet through-holes on the insert; and sealing acenter through-hole, branched channels, and aliquoting through-holesdisposed on the insert when the rivets are inserted into the rivetthrough-holes.

In yet another variant, a method for creating holes in an MA chip,comprises inserting an MA chip between a top and a bottom of anenclosure; aligning the MA chip directly above a reverse mold in theenclosure; inserting a plurality of pins attached to a bottom of a pinhead into a plurality of tapered holes on the top of the enclosure; andpressing a top of the pin head so that the MA chip is pushed into thereverse mold. The MA chip and enclosure are aligned when the reversemold has a pattern that matches a pattern on the MA chip and the reversemold has a flattened edge that matches a flattened edge on theenclosure.

In another variant, a method for mass production of MA chips, comprisesconducting photolithography to produce a silicon mold; injectingPolydimethylsiloxane (PDMS) into the silicon mold; heating the siliconmold containing the PDMS to produce a first layer of an MA chip havingchannels and an inlet well; cutting plastic material with a laser toproduce a second layer of an MA chip having a well array and channels;and aligning the first layer directly on top of the second layer so thechannels on both layers overlap. The channels on the first layer arelonger and narrower than the channels on the second layer.

In a further variant, a method for mass production of MA chips,comprises cutting plastic material with a laser to produce a first layerof an MA chip having a well array and an inlet well; conductingphotolithography to produce a silicon mold; injectingPolydimethylsiloxane (PDMS) into the silicon mold; heating the siliconmold containing the PDMS to produce a second layer of an MA chip havingradial channels and an alignment mark array; and aligning the firstlayer directly on top of the second layer so the well array and thealignment mark array overlap.

In yet another variant, a method for making MA chips, comprises cuttingplastic material with a laser to produce a first layer of an MA chiphaving an inlet well, outlet well array, main-channel array, andsub-channel array; adhering a second membrane layer on top of the firstlayer using an adhesive on the second membrane layer; cutting an area ofthe membrane directly on top of the inlet well with a puncher; andsecuring a PDMS cap on top of the exposed inlet well. The cap isconfigured to receive liquid, which distributes from the cap to theinlet well, the main channels, the sub-channels, and finally the outletwells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bMA Chip Type 1.

FIG. 1B is a magnified portion of the MA chip in FIG. 1A.

FIG. 1C is a magnified portion of an MA chip with four segments.

FIG. 2A is a magnified portion of a first segment of an MA chip.

FIG. 2B is a magnified portion of a second segment of an MA chip.

FIG. 2C is a magnified portion of a third segment of an MA chip.

FIG. 2D is a magnified portion of a second segment of an MA chip.

FIG. 3A is a diagram of a bMA Chip Type 2.

FIG. 3B is a magnified portion of a bMA Chip Type 2.

FIG. 3C is a bMA Chip Type 3.

FIG. 3D is a magnified portion of a bMA Chip Type 3.

FIG. 4A is an MA chip insert.

FIG. 4B is an MA chip base.

FIG. 4C is a method for making an MA chip insert.

FIG. 4D is a method for making an MA chip base.

FIG. 4E is the insert and base prior to being assembled together.

FIG. 4F is the insert and base after the process of assembling themtogether as described in the methods of FIGS. 4C and 4D.

FIG. 5 is a multiplex hole punch.

FIG. 5A is an MA Chip top and bottom enclosure assembly.

FIG. 5B is an MA Chip with microchannel array.

FIG. 5C is a reverse mold of an MA chip.

FIG. 5D is an assembled enclosure of an MA chip without the top.

FIG. 5E is a complete assembled enclosure of an MA Chip.

FIG. 6A is an MA chip assembly with long and narrow channels on a toplayer.

FIG. 6B is an MA chip assembly with short and wide channels on a bottomlayer.

FIG. 6C is an MA chip assembly with the top and bottom layer combined.

FIG. 6D is an MA chip assembly with short and wide channels on a toplayer.

FIG. 6E is an MA chip assembly with long and narrow channels on a bottomlayer.

FIG. 6F is an MA chip assembly with the top and bottom layer combined.

FIG. 7 is a side view diagram of the rMA chip.

FIG. 8 is a top view diagram of the rMA chip.

FIG. 8A is a method of assembling the rMA chip.

FIG. 9A is an rMA chip before loading liquid into the chip.

FIG. 9B is an rMA chip after loading liquid into the chip.

FIG. 9C is an rMA chip after removing the tape.

FIG. 10A is a design of an rMA chip.

FIG. 10B is a magnified portion of an rMA chip.

FIG. 10C is a prototype of a PMMA rMA chip.

DETAILED DESCRIPTION

The following Reference Numbers are used in this document:

-   100 Microfluidic Aliquoting (MA) chip-   110 Inlet well-   112 Plurality of channels-   114 Plurality of outlet wells-   114 a First segments-   114 b Second segments-   114 c Third segments-   114 d Fourth segments-   116 Width of first segments-   118 Width of second segments-   122 Width of third segments-   126 Top surface of MA chip-   128 Identifying millimeter-scale number-   129 Sealed flow channel-   130 MA Chip Base-   131 Aliquoting through-holes-   132 Liquid reservoir wells-   133 Open flow channel-   134 Joining rivet-   135 Plurality of rivet through-holes-   136 Engraved identification number-   137 Center through-hole-   138 Flattened edge-   139 Insert-   140 Multiplex hole punch pin head-   141 Sink Plateau surface-   142 Top enclosure of multiplex hole punch-   143 Pins-   144 Reverse mold of MA chip-   146 Bottom enclosure of multiplex hole punch-   148 MA chip with microchannel array-   150 Bottom surface of MA chip-   152 Impermeable membrane-   154 Well filled with liquid-   156 rMA Chip-   158 rMA Chip with liquid-   160 384 well rMA Chip    Section 1: Branched Microfluidic Aliquot Chip Type 1 (bMA-Chip T1)

In a variant, referring generally to FIGS. 1A-1C, the bMA-Chip Type 1comprises a chip 100 having a center, a top surface 126, a bottomsurface 150, an outer edge and a thickness. The chip 100 has an inletwell 110 disposed substantially at the center of the chip 100, extendinginto and accessible from the top surface 126 of the chip 100. Aplurality of outlet wells 114 are disposed in an outer portion of thechip 100, extending into and accessible from the top surface 126 of thechip 100. A plurality of multiple segments 112 extend from the inletwell 110 to the outlet wells 114, wherein the multiple segments 112comprise branched channels. A plurality of first segments 114 a form aninner section, a plurality of last segments form an outer section, and aplurality of segments between the first segments and the last segmentsform a middle section. The first segment 114 a is connected to the inletwell 110 in a radial pattern and each segment after the first segment114 a is divided from a prior segment in a radial pattern. The pluralityof segments in the middle section form a curved portion generally in theshape of a bend, whereby one of the first segments 114 a is joined tothe bend of a segment in the middle section. A first channel of a lastsegment is shorter than a second channel of the last segment. Everyother outlet well 114 is disposed farther away from the inlet well 110than an adjacent outlet well 114.

In another variant, the multiple segments 112 connect the inlet well 110to 96 outlet wells 114. The number of channels in each segment, from theinside section to the outside section, is 24, 48, and 96, respectively.The bMA-Chip T1 can also contain four segments: the 1st segment 114 a(inside section), the 2nd 114 b and 3rd segments 114 c (middlesections), and the 4th segment 114 d (outside section). The number ofchannels in each segment, from the inside section to the outsidesection, is 12, 24, 48, and 96 channels, respectively. The bMA-Chip T1can also contain other segments, such as 2, 5, and 6. The liquid andcells can be uniformly distributed into 96 outlet wells 114 through themultiple segments 112 of bMA-Chip T1. The chip 100 is configured tomaintain uniform distribution of a liquid and cells from the inlet wellto the outlet wells.

Referring generally to FIGS. 2A-2D, in a further variant, the distancebetween the segments vary. The first segments 114 a have a width 116 of125 μm. The second segments 114 b have a width 118 of 75 μm. The thirdsegments 114 c have a width 122 of 50 μm. When there are 4 segments, thefirst segments 114 a have a width 116 of 150 μm, second segments 114 bhave a width 118 of 125 μm, third segments 114 c have a width 122 of 75μm, and fourth segments 114 d have a width of 50 μm.

Section 2: Branched Microfluidic Aliquot Chip Type 2 (bMA-Chip T2)

In a variant, referring to FIGS. 3A-3B, the bMA Chip-T2 comprises fourfirst segments 114 a that extend outward from the inlet well 110 in fourcardinal directions, respectively. Each segment branches at a 90 degreeangle in two opposite directions into another segment forming a set ofbranched channels. Each set of branched channels on a last segmentcorrespond to a set of outlet wells 114. The chip also comprises a thinsheet of flexible or semi-rigid material such as, polydimethylsiloxane(PDMS), PS (polystyrene) PC (polycarbonate), and PMMA (poly (methylmethacrylate)) with a thickness of approximately 1 mm. The bMA Chip-T2has the overall form of a square, having a geometric center and a sideof approximately 6 cm. The bMA Chip-T2 is sized and shaped to fit in aPetri dish having a diameter of approximately 8.5-10 cm. The sheetforming the bMA Chip-T2 has a top surface and a bottom surface,corresponding to the top 126 and bottom 150 surfaces of the overall bMAChip-T2, respectively. The inlet well has a diameter of approximately2-4 mm. The inlet well has a volume of 3-5 μl. The inlet well isdisposed at the geometric center of the bMA Chip-T2. The inlet well isaccessible to a user from the top surface 126 of the bMA Chip-T2, forloading a cell suspension into the bMA Chip-T2.

In another variant, the outlet wells 114 are in the form of round holesextending completely through the bMA Chip-T2. The outlet wells 114 maybe in the shape of an oval, triangle, square, rectangle, rhombus,trapezoid, or pentagon. The outlet wells 114 have a diameter of 1.5-2mm. The outlet wells 114 each have a volume of 1-3 μl. The outlet wells114 are distributed along four cardinal directions of the bMA Chip-T2.The outlet wells 114 are accessible to a user from the top surface 126of the bMA Chip-T2, for retrieving isolated cells from the bMA Chip-T2.

In a further variant, 16 outlet wells 114 are arranged into a set ofbranched channels, corresponding to a total of 64 outlet wells 114arranged into four sets of branched channels. All channels have a widthof approximately 50 μm. Each set of branched channels consists of foursegments that are connected to 16 outlet wells 114. The total number ofbranched channels in each set from the 1st segment 114 a to the 4thsegment 114 d is 2, 4, 8, and 16, respectively. Relatively smallμm-scale markings are disposed inside the outlet wells 114 foridentifying the outlet wells 114 under microscopic observation, andrelatively large mm-scale markings 128 are disposed outside of the 1stsegment 114 a along four cardinal directions.

Section 3: Branched Microfluidic Aliquot Chip Type 3 (bMA-Chip T3)

In a variant, referring to FIGS. 3C-3D, the bMA Chip-T3 comprises aplurality of first segments 114 a that extend outward radially from theinlet well 110. Each segment branches at a 90 degree angle in twoopposite directions into another segment forming a set of branchedchannels. Each set of branched channels on a last segment correspond toa set of outlet wells 114. The chip further comprises a thin sheet offlexible or semi-rigid material such as, polydimethylsiloxane (PDMS), PS(polystyrene) PC (polycarbonate), and PMMA (poly (methyl methacrylate))with a thickness of approximately 1 mm. The bMA Chip-T3 has the overallform of a disk, having a geometric center and a diameter ofapproximately 8 cm. The bMA Chip-T3 is sized and shaped to fit in aPetri dish having a diameter of approximately 8.5-10 cm. The sheetforming the bMA Chip-T3 has a top surface and a bottom surface,corresponding to the top 126 and bottom 150 surfaces of the overall bMAChip-T3, respectively.

In another variant, the inlet well 110 has a diameter of approximately2-4 mm and a volume of 3-5 μl. The inlet well 110 is disposed at thegeometric center of the bMA Chip-T3 and is accessible to a user from thetop surface of the bMA Chip-T3, for loading a cell suspension into thebMA Chip-T3. The outlet wells 114 are in the form of round holesextending completely through the bMA Chip-T3. The outlet wells 114 maybe in the shape of an oval, triangle, square, rectangle, rhombus,trapezoid, or pentagon. The outlet wells 114 have a diameter ofapproximately 1.5-2 mm and a volume of 1-3 μl. The outlet wells 114 aredistributed around an outer annular portion of the bMA Chip-T3 with thebranched channels. The outlet wells 114 are accessible to a user fromthe top surface of the bMA Chip-T3, for retrieving isolated cells fromthe bMA Chip-T3.

In a further variant, four outlet wells 114 are arranged into a set ofbranched channels, corresponding to a total of 64 outlet wells 114arranged into 16 sets of branched channels. All channels have a width ofapproximately 50 μm. Each set of branched channels consists of twosegments that are connected to 4 total outlet wells 114. The totalnumber of branched channels for the 1st segment 114 a and the 2ndsegment 114 b is 32 and 64, respectively.

In yet another variant, relatively large mm-scale markings 128 aredisposed outside of the outlet wells 114 for identifying the outletwells 114 under naked-eye observation. The markings 128 may be otherthan numbers or letters, such as 1D and 2D barcodes for identifying theoutlet wells 114 by using an imaging software. For the linear 1Dbarcodes, the information is stored in the relationship of the widths ofthe bars (spaces) to each other. For the stacked 2D barcodes, severalstacked linear barcodes are used to encode the information. Compared tostacked barcodes the information of the matrix 2D barcodes is not storedby using different bar (space) widths. Instead the position of black orwhite dots is relevant.

Section 4: Injection Mold Design

In a variant, referring to FIG. 4A, an MA chip insert 139 for making anMA chip 100, comprises a center through-hole 137, a plurality ofaliquoting through-holes 131, a plurality of branched channels, aplurality of outlet wells 114, a bottom surface, a top surface, aflattened edge 138, and a plurality of rivet through-holes 135. Theinsert 139 has a design configured to match a design of an MA chip 100.The rivet through-holes 135 are placed in a pattern on the insert 139illustrated in FIG. 4A, and correspond in spacing to correspondingrivets 134 on the base 130 in FIG. 4B.

In another variant, referring to FIG. 4B, an MA chip base 130 for makingan MA chip 100, comprises a bottom surface having bottom pillars, a topsurface having edge wells, a sink plateau surface 141 having a flattenededge 138, and a plurality of rivets 134.

In a further variant, referring FIGS. 4C and 4D, a method for making anMA chip 100 is illustrated. The method comprises inserting a first pairof patterned metal injection molds into an injection molding machine.Thermoplastic is injected into the first pair of patterned metalinjection molds. Demolding is done to produce an insert 139 having areverse pattern of the first pair of molds. A second pair of patternedmetal injection molds is inserted into the injection molding machine.Thermoplastic is inserted into the second pair of patterned metalinjection molds. Demolding is done to produce a base 130 having areverse pattern of the second pair of molds. A plurality of rivets 134are inserted on the base 130 into a plurality of rivet through-holes 135on the insert 139. When the rivets 134 are inserted into the rivetthrough-holes 135, a center through-hole 137, branched channels, andaliquoting through-holes 131 are sealed and disposed onto the insert139. FIGS. 4E and 4F illustrate before and after, respectively, theprocess of assembling the insert 139 and base together 130 as describedin the process of FIGS. 4C and 4D. FIG. 4E illustrates a centerthrough-hole 137 which converts into an inlet well 110 in FIG. 4F, analiquoting through-hole 131 which converts into an outlet well 114 inFIG. 4F, and an open flow channel 133 which converts into a sealed flowchannel 129 in FIG. 4F.

In another variant, the branched channel design provides large spacing(>0.4 mm) between the channels. The large space allows a channel-sealingmechanism between the insert 139 and base 130 by inserting rivets 134into the rivet through-holes 135. The flattened edge 138 is added toensure the alignment between the MA-Chip insert 139 and base 130. The 24liquid reservoir wells 132 are designed for carrying buffers, culturemediums, or cell suspension. Each well 132 can contain 30 to 400 μlvolume of liquid. The aliquot cells can be transferred to the edge wellsfor long term culture and cell expansion. The wells can serve as amedium reservoir during on-chip tissue culture to prevent culture mediumevaporation. Each well is assigned with an alphabet as a wellidentification method 136. The well identification alphabets 136 arepositively engraved on the top. The patterned bottom surface preventsthe viewing area from scratches when it lays down by providing a smallgap between the bottom surface and the rough surface. A patterned sinkplateau surface 141 exactly matches the MA-Chip insert 139 by using theflattened edge 138 for perfect alignment. The identification numbers foraliquot wells are engraved on the base 130 allowing large spacingbetween MA-Chip channels. The large spacing between MA-Chip channelsprovide better manufacturability.

Section 5: Multiplex Hole Puncher Method

In a variant, referring to FIGS. 5, and 5A-5E, a method for creatingholes in an MA chip 100 using a multiplex hole puncher, comprisesinserting an MA chip 100 between a top 142 and a bottom 146 of anenclosure; aligning the MA chip 100 directly above a reverse mold 144 inthe enclosure; inserting a plurality of pins 143 attached to a bottom ofa pin head 140 into a plurality of tapered holes on the top 142 of theenclosure; and pressing a top of the pin head 140 so that the MA chip100 is pushed into the reverse mold 144. The MA chip 100 and enclosureare aligned when the reverse mold 144 has a pattern that matches apattern on the MA chip 100 and the reverse mold 144 has a flattened edge138 that matches a flattened edge on the enclosure.

In another variant, the position of the pins 143 is matched with theposition of the MA-Chip wells to punch the MA-Chip holes at the sametime. This design reduces the hole punching process time. Hollow metalalloy punch pins 143 are secured on the rigid metal alloy substrate suchas stainless steel or brass. The metal substrate holds the pins 143 bytapered hole, which allows replacement of the pin 143. The through-holesguide the punch pins 143 to the exact position of the MA-Chip well. Theshape of the bottom sink plateau with a flattened edge 138 matches thereverse mold 144 of the MA-Chip 100. The flattened edge 138 is used toalign the reverse mold 144 to the top 142 enclosure. The top 142 andbottom 146 enclosures are mirror images. The patterned surface of thereverse mold 144 PDMS block matches the channel and well design of theMA-Chip 100 and thus, ensures the alignment between the MA-Chip holesand channels. The radial shape with a flattened edge 138 matches thesink plateau in the top 142 and bottom 146 enclosures. The flattenededge 138 is used to automatically align the reverse mold 144 to theenclosure.

Section 6: Method of Mass-Producing an MA Chip

In a variant, referring to FIGS. 6A-6F, a method for mass production ofMA chips 100, comprises conducting photolithography to produce a siliconmold; injecting Polydimethylsiloxane (PDMS) into the silicon mold;heating the silicon mold containing the PDMS to produce a first layer ofan MA chip 100 having channels and an inlet well 110; cutting plasticmaterial with a laser to produce a second layer of an MA chip 100 havinga well array and channels; and aligning the first layer directly on topof the second layer so the channels on both layers overlap. The channelson the first layer are longer and narrower than the channels on thesecond layer.

In another variant, a method for mass production of MA chips 100,comprises cutting plastic material with a laser to produce a first layerof an MA chip 100 having a well array and an inlet well 110; conductingphotolithography to produce a silicon mold; injectingPolydimethylsiloxane (PDMS) into the silicon mold; heating the siliconmold containing the PDMS to produce a second layer of an MA chip 100having radial channels and an alignment mark array; and aligning thefirst layer directly on top of the second layer so the well array andthe alignment mark array overlap.

In a further variant, the assembled MA-Chip 100 is made of two patternedlayers. The top layer is made of PDMS by photolithography and containslong and narrow channels (2-4 cm in length and 0.03-0.1 mm in width) anda central through-hole (2-4 mm in diameter). The bottom layer is made ofplastic materials by laser cutting or injection molding, such as PS, PP,PMMA, and PC, and contains a well array (1-2 mm in diameter) andassociated short and wide channels (0.5-2.5 mm in length and 0.3-0.5 mmin width). When the two patterned layers are assembled and bonded, thelong and narrow channels and short and wide channels can be welloverlapped for uniform liquid distribution. A microliter of liquid,typically between 100 μL and 200 μL, can be injected into the inlet well110 by a pipette and uniformly dispensed into 100 open wells.

Section 7: Rectangular MA Chip

In a variant, referring to FIGS. 7-10C, a method for making MA chips100, comprises cutting plastic material with a laser to produce a firstlayer of an MA chip 100 having an inlet well 110, outlet well array,main-channel array, and sub-channel array; adhering a second membranelayer 152 on top of the first layer using an adhesive on the secondmembrane layer 152; cutting an area of the membrane 152 directly on topof the inlet well 110 with a puncher; and securing a PDMS cap on top ofthe exposed inlet well 110. The cap is configured to receive liquid,which distributes from the cap to the inlet well 110, the main channels,the sub-channels, and finally the outlet wells 114, as illustrated inFIG. 8A.

In another variant, a rectangular MA-Chip (rMA-Chip) 156 where alloutlet wells 114 are patterned in a rectangular array. The top of thewell array is connected to the channel array and both of them are sealedby a gas-permeable and liquid-impermeable membrane 152. Liquids areloaded with pipettes or syringes and will flow into the channel arrayand then into the well array by continuously pushing the air to theoutside. After loading the liquid, the membrane 152 is removed and thewell array with liquid 154 inside is obtained. After removing themembrane 152, the wells are open to the air and the liquids associatedwith single cells may be confirmed by microscope and then retrieved witha pipette.

In a further variant, the rMA-Chip 156 is rectangular in shape and127.8±5 mm in length, 85.5±3 mm in width, and 1-10 mm in height. TherMA-Chip 156 has two layers: the top layer and the bottom layer. The toplayer is a membrane 152 with a thickness of 0.03-0.3 mm. The top layeris gas-permeable and liquid-impermeable. The top layer is biocompatibleand not harmful to cells. The top layer is transparent and flexible.

In yet another variant, the outlet wells 114 are 0.5-5 mm in diameterand 1-10 mm in height. The number of outlet wells 114 in one rMA-Chipcan be 32, 64 (32×2), 96 (32×4), 384 (96×4) 160, or 1536 (384×4). Outletwells 114 can be in other shapes such as a rectangle, triangle, or oval.Outlet wells 114 are uniformly distributed throughout the rMA-Chip 156.Each outlet well 114 is labeled by a micro-scale number and letterdesigned for microscopic observation and macro-scale number and letterdesigned for naked-eye observation.

What is claimed is:
 1. A method for making an MA chip, comprising:inserting a first pair of patterned metal injection molds into aninjection molding machine; injecting thermoplastic into the first pairof patterned metal injection molds; demolding to produce an inserthaving a reverse pattern of the first pair of molds; inserting a secondpair of patterned metal injection molds into the injection moldingmachine; inserting thermoplastic into the second pair of patterned metalinjection molds; demolding to produce a base having a reverse pattern ofthe second pair of molds; inserting a plurality of rivets on the baseinto a plurality of rivet through-holes on the insert; and sealing acenter through-hole, branched channels, and aliquoting through-holesdisposed on the insert when the rivets are inserted into the rivetthrough-holes.